Compositions and Methods for Inhibiting Expression of an RNA from West Nile Virus

ABSTRACT

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of an RNA from the West Nile virus (WNV), and the use of the dsRNA to treat pathological processes mediated by WNV infection, such as viral encephalitis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/122,659, which is a National Stage of International Application No.PCT/US2009/059737, filed on Oct. 6, 2009, which claims the benefit ofU.S. Provisional Application No. 61/102,976, filed on Oct. 6, 2008. Allof the prior applications are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the U.S. government under grant number 1 U01 AI075419-01, awardedby the Department of Health and Human Services at the NationalInstitutes of Health. The government has certain rights in theinvention.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 24733US_sequencelisting.txt, created on Oct. 22, 2013,with a size of 20,000 bytes. The sequence listing is incorporated byreference.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of anRNA from the West Nile virus (WNV), and the use of the dsRNA to treatpathological processes mediated by WNV infection, such as viralencephalitis.

BACKGROUND OF THE INVENTION

The West Nile virus (WNV) is a mosquito-born flaviviruses that can causeacute neurological illness with up to 30% mortality and permanentneurological disabilities in the survivors. The virus has beenidentified by the National Institute of Allergy and Infectious Diseases(NIAID) as a category B bioterrorism agent. There is currently noeffective treatment for WNV infection.

Flaviviruses are small (40-60 nm) enveloped viruses with asingle-stranded positive-sense RNA genome that is approximately 11 kblong. The genome encodes a long polyprotein and is flanked by twonontranslated regions (5′ and 3′ NTR). The 3000 amino acid polyproteinis cleaved into three structural proteins, the core protein C, thepremembrane protein (prM) and the envelope protein (E) and sevennonstructural (NS) viral proteins, NS1-NS5. Several regions in the viralgenome are highly conserved among the different strains within a speciesas well as across the different flaviviral species. These include, inparticular, the region coding for the cd loop of the envelope protein,and the region coding for the nonstructural proteins NS3 and NS5. Allflaviviruses share two short conserved RNA sequences near the 3′ end ofthe RNA genome. For mosquito-borne flaviviruses, such as WNV, theelements are the 26 nt CS1 and the 24 nt CS2. Part of CS1 iscomplementary to a conserved sequence near the 5′ end of the genome inthe region encoding the capsid protein which is referred to as 5′CS.Base pairing of these sequences is thought to be involved in cyclizationof the viral genome.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of an RNA.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods, for inhibiting the expression of an RNA ofWest Nile Virus (WNV). The invention also provides compositions andmethods for treating pathological conditions and diseases caused by WNVinfection, such as encephalitis. The dsRNA featured in the inventioncomprises an RNA strand (the antisense strand) having a region that isless than 30 nucleotides in length, generally 19-24 nucleotides inlength, and is substantially complementary to at least part of an mRNAtranscript of an RNA from WNV.

In one embodiment, the invention provides dsRNA molecules for inhibitingthe expression of an RNA of WNV and viral replication. The dsRNAincludes at least two sequences that are complementary to each other.The dsRNA includes a sense strand having a first sequence and anantisense strand having a second sequence. The antisense strand includesa nucleotide sequence that is substantially complementary to at leastpart of an mRNA encoded by WNV, and the region of complementarity isless than 30 nucleotides in length, generally 19-24 nucleotides inlength. The dsRNA, upon contacting with a cell infected with WNV,inhibits the expression of an RNA from WNV by at least 40%.

For example, the dsRNA molecules featured in the invention can include afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Table 2 and the second sequence is selectedfrom the group consisting of the antisense sequences of Table 2. ThedsRNA molecules featured in the invention can include naturallyoccurring nucleotides or can include at least one modified nucleotide,such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative. Alternatively, the modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro-modified-nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. The dsRNA molecules featured in the invention can includenaturally occurring backbone linkages, e.g., phosphodiester linkages orcan include at least one non phosphodiester linkage, such asphosphorothioate, phosphorodithioate, phosphoramidate, phosphonate andalkyl-phosphonate backbone linkage. Generally, such modified sequencewill be based on a first sequence of said dsRNA selected from the groupconsisting of the sense sequences of Table 2 and a second sequenceselected from the group consisting of the antisense sequences of Table2.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs featured in the invention. The cell is generally a mammaliancell, such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the replication of WNV in an organism,generally a human subject, comprising one or more of the dsRNA featuredin the invention and a pharmaceutically acceptable carrier or deliveryvehicle.

In another embodiment, the invention provides a method for inhibitingthe expression of an RNA of WNV in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA includes a sense        strand having a first sequence and an antisense strand having a        second sequence. The antisense strand includes a region of        complementarity that is substantially complementary to at least        a part of an mRNA encoded by WNV, and wherein the region of        complementarity is less than 30 nucleotides in length, generally        19-24 nucleotides in length, and wherein the dsRNA, upon contact        with a cell infected with WNV inhibits expression of an RNA from        WNV by at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of WNV,        thereby inhibiting expression of an RNA from WNV in the cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes mediated by WNV infection,such as encephalitis, comprising administering to a patient in need ofsuch treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs featuredin the invention.

In another embodiment, the invention provides vectors for inhibiting theexpression of an RNA of WNV in a cell, where the vector includes aregulatory sequence operably linked to a nucleotide sequence thatencodes at least one strand of one of the dsRNAs featured in theinvention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of an RNA of WNV in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA featured in theinvention.

In one aspect, the invention provides a method for delivering a nucleicacid drug, e.g., a dsRNA, to a subject. The subject can be a mammal,such as a human or non-human primate. Delivery can be, for example, bylocalized delivery, e.g., injection or infusion, into the brain, such asinto white matter of the brain, e.g., into the corpus callosum. Inanother embodiment, the dsRNA is delivered by intrastriatal infusion,into the striatum, such as into the corpus striatum. In otherembodiments, delivery of the dsRNA is systemic, such as by intravenousor intramuscular administration, by administration to target cellsex-planted from the patient followed by reintroduction into the patient,or by any other means that allows for introduction into a desired targetcell.

In one embodiment, the nucleic acid drug is a double-strandedribonucleic acid (dsRNA) molecule for inhibiting the expression of oneof the genes of WNV and for inhibiting viral replication. The dsRNA caninclude at least two sequences that are complementary to each other. ThedsRNA can include a sense strand comprising a first sequence and anantisense strand comprising a second sequence. The antisense strandcomprises a nucleotide sequence which is substantially complementary toat least part of an mRNA encoded by WNV, and the region ofcomplementarity is preferably less than 30 nucleotides in length,generally 19-24 nucleotides in length. In one embodiment, the dsRNA,when evaluated in an in vitro assay described herein, inhibitsexpression of an RNA of WNV by at least 40%.

For example, the dsRNA molecules can include a first sequence that isselected from the group consisting of the sense sequences of Table 2,and a second sequence selected from the group consisting of theantisense sequences of Table 2. The dsRNA molecules can includenaturally occurring nucleotides or can include at least one modifiednucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotidehaving a 5′-phosphorothioate group, or a terminal nucleotide linked to acholesteryl derivative. Alternatively, the modified nucleotide may bechosen from the group consisting of a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modifiednucleotide, morpholino nucleotide, a phosphoramidate, and a non-naturalbase comprising nucleotide. The dsRNA molecules featured in theinvention can include naturally occurring backbone linkages, e.g.,phosphodiester linkages or can include at least one non phosphodiesterlinkage, such as phosphorothioate, phosphorodithioate, phosphoramidate,phosphonate and alkyl-phosphonate backbone linkage. Generally, suchmodified sequences will be based on a first sequence of a dsRNA selectedfrom the group consisting of the sense sequences of Table 2, and asecond sequence selected from the group consisting of the antisensesequences of Table 2.

In one embodiment, the dsRNA targets the part of the mRNA encoding thecore protein C, the premembrane protein (prM), the envelope protein (E),the capsid protein, or one of the nonstructural proteins, NS1, NS2(e.g., NS2a or NS2b), NS3, NS4, or NS5. In another embodiment, the dsRNAtargets a region of WNV mRNA that overlaps two separate protein-codingregions. In yet another embodiment, more than one type of dsRNA isadministered, but only one distinct protein-coding region is targeted.In another embodiment, more than one type of dsRNA is administered, eachtargeting a distinct protein-coding region. In another embodiment, thedsRNA targets one of the conserved sequences near the 3′ end of the RNAgenome (CS1 or CS2), or a conserved sequence at the 5′ end of the genomein the region encoding the capsid protein, called 5′CS. In anotherembodiment, the dsRNA targets a region that is conserved with otherflaviviruses, such as the region of the mRNA that encodes the cd loop ofthe envelope protein, or a region of NS3.

In one embodiment, the dsRNA does not activate the immune system, e.g.,it does not increase cytokine levels, such as TNF-alpha or IFN-alphalevels. For example, when measured by an assay, such as an in vitro PBMCassay, such as described herein, the increase in levels of TNF-alpha orIFN-alpha, is less than 30%, 20%, or 10% of control cells treated with acontrol dsRNA, such as a dsRNA that does not target a WNV mRNA.

In another aspect, the invention provides a method for inhibiting theexpression of a WNV mRNA in a subject. The subject can be a mammal, suchas a human or non-human primate.

In one embodiment, the nucleic acid drug is a double-strandedribonucleic acid (dsRNA) molecule for inhibiting the expression of anRNA of WNV and for inhibiting viral replication. The dsRNA can includeat least two sequences that are complementary to each other. The dsRNAcan include a sense strand having a first sequence and an antisensestrand having a second sequence. The antisense strand includes anucleotide sequence that is substantially complementary to at least partof an mRNA encoded by an RNA from WNV, and the region of complementarityis preferably less than 30 nucleotides in length, generally 19-24nucleotides in length. In one embodiment, the dsRNA, when evaluated inan in vitro assay described herein, inhibits expression of an RNA fromWNV by at least 40%.

In another aspect, the invention provides a method for treating,preventing, delaying or managing a pathological process or symptommediated by WNV infection, such as encephalitis, in a subject. Thesubject can be a mammal, such as a human or non-human primate. Themethod includes delivery, e.g., localized delivery, such as by injectionor infusion, of a therapeutic amount of a nucleic acid drug, e.g., adsRNA. In some embodiments, delivery of the dsRNA is systemic, such asby intravenous or intramuscular administration, by administration totarget cells ex-planted from the patient followed by reintroduction intothe patient, or by any other means that allows for introduction into adesired target cell. In other embodiments, delivery is into the brain,such as into the white matter of the brain, such as into the corpuscallosum of the subject. In one embodiment, the method provides deliveryto oligodendrocytes by infusion into the corpus callosum (e.g., byintracallosal infusion).

In another aspect, the invention provides a pharmaceutical compositionfor inhibiting the replication of WNV in an organism, generally a humansubject, containing one or more of the dsRNA featured in the inventionand a pharmaceutically acceptable carrier or delivery vehicle.

In another aspect, the invention provides vectors for inhibiting theexpression of an RNA of WNV in a cell, where the vector includes aregulatory sequence operably linked to a nucleotide sequence thatencodes at least one strand of one of the dsRNAs featured in theinvention.

In another embodiment, the invention provides a cell containing a vectorfor inhibiting the expression of an RNA of WNV in a cell. The vectorincludes a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNAs featured in theinvention.

In other embodiments, the invention provides lipid formulations of thedsRNAs described, e.g., in Table 2. Exemplary siRNA-lipid formulationsare described herein.

The details of one or more embodiments featured in the invention are setforth in the description below. Other features, objects, and advantagesfeatured in the invention will be apparent from the description and fromthe claims.

BRIEF DESCRIPTION OF THE FIGURES

No figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of an RNA fromWNV in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by WNV infection using dsRNA. dsRNA directsthe sequence-specific degradation of mRNA through a process known as RNAinterference (RNAi).

The dsRNA suitable for use in the methods described herein include anRNA strand (the antisense strand) having a region that is less than 30nucleotides in length, generally 19-24 nucleotides in length, and thatis substantially complementary to at least part of an mRNA transcript ofan RNA from WNV. The use of these dsRNAs enables the targeteddegradation of mRNAs of genes that are implicated in replication and ormaintenance of WNV infection and the occurrence of neurologicalsymptoms, such as encephalitis, in a subject infected with WNV. Usingcell-based and animal assays, the present inventors have demonstratedthat very low dosages of these dsRNA can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of anRNA from WNV. Thus, the methods and compositions featured herein includedsRNAs useful for treating pathological processes mediated by WNVinfection, such as encephalitis, by targeting an RNA involved in WNVreplication and/or maintainance in a cell.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of anRNA from WNV, as well as compositions and methods for treating diseasesand disorders caused by infection with WNV, such as encephalitis. Thepharmaceutical compositions suitable for use in the featured methodsinclude a dsRNA having an antisense strand with a region ofcomplementarity that is less than 30 nucleotides in length, generally19-24 nucleotides in length, and is substantially complementary to atleast part of an RNA transcript of an RNA from WNV, together with apharmaceutically acceptable carrier.

Accordingly, certain aspects featured in the invention providepharmaceutical compositions containing the dsRNAs described hereintogether with a pharmaceutically acceptable carrier, methods of usingthe compositions to inhibit expression of an RNA from WNV, and methodsof using the pharmaceutical compositions to treat diseases caused byinfection with WNV.

When the organism to be treated is a mammal such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection. In other embodiments, the compositions areadministered by intracranial infusion or injection.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, “WNV” refers to the WNV strain B 956 having a referencesequence AY532665. Other WNV strains are suitable for use in the methodsand compositions featured in the invention. Accession numbers of otherWNV sequences include NC_(—)009942, NC_(—)001563, AM404308, EF429200,EF429199, EF429198, EF429197, EF657887, EF530047, EU155484, EU081844,EF571854, DQ256376, DQ176636, DQ176637, DQ211652, AY765264, DQ318019,DQ318020, DQ164206, DQ164205, DQ164204, DQ164203, DQ164198, DQ164197,DQ164196, AY848696, AY848695, AY848697, AY842931, DQ066423, AY688948,AY603654, AY639643, AY639642, AY639641, AY639640, AY289214, AY660002,AY532665, AY490240, AF481864, AF196835, AF260969, AF260968, AF260967,and AF206518.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an RNA from WNV, including mRNA that is a product of RNA processingof a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidehaving the first nucleotide sequence to the oligonucleotide orpolynucleotide having the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA having one oligonucleotide 21 nucleotides in length andanother oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., encoding WNV)) including a 5′ UTR, an open reading frame(ORF), or a 3′ UTR. For example, a polynucleotide is complementary to atleast a part of a WNV mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding WNV.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. In general, the majority of nucleotides ofeach strand are ribonucleotides, but as described in detail herein, eachor both strands can also include at least one non-ribonucleotide, e.g.,a deoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′ end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′ end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. dsRNAs as used herein are also referred to as“siRNAs” (short interfering RNAs).

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA thatincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” where the cell is part of aliving organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electro-poration and lipofection.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to an RNA from WNV, herein refer to the at least partialsuppression of the expression of an RNA from WNV, as manifested by areduction of the amount of mRNA transcribed from the positive strand RNAfrom WNV which may be isolated from a first cell or group of cells inwhich an RNA from WNV is transcribed and which has or have been treatedsuch that the expression of an mRNA from WNV is inhibited, as comparedto a second cell or group of cells substantially identical to the firstcell or group of cells but which has or have not been so treated(control cells). The degree of inhibition is usually expressed in termsof

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to WNV genometranscription, e.g., the amount of protein encoded by an RNA from WNV,or the number of cells displaying a certain phenotype, e.g infectionwith WNV. In principle, WNV RNA silencing may be determined in any cellexpressing the target, either constitutively or by genomic engineering,and by any appropriate assay. However, when a reference is needed inorder to determine whether a given dsRNA inhibits the expression of anRNA from WNV by a certain degree and therefore is encompassed by theinstant invention, an assay described herein, or an assay known in theart shall serve as such reference.

For example, in certain instances, expression of an RNA from WNV issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of the double-stranded oligonucleotide. In someembodiments, an RNA from WNV is suppressed by at least about 60%, 70%,or 80% by administration of the double-stranded oligonucleotide. In someembodiments, an RNA from WNV is suppressed by at least about 85%, 90%,or 95% by administration of the double-stranded oligonucleotide. WNVmRNA levels can be assayed, for example, by branched-DNA assay or byRT-PCR. In an alternative approach, protein levels can be assayed, forexample, by Western blot, or ELISA, or other common methods. In yetother alternatives, the efficacy of a dsRNA targeting WNV can be assayedby its ability to inhibit WNV replication in cell culture, or by itsability to protect mice from WNV-induced encephalitis.

As used herein in the context of WNV expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by WNV infection. In the context of thepresent invention insofar as it relates to any of the other conditionsrecited herein below (other than pathological processes mediated by WNVexpression), the terms “treat,” “treatment,” and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression of such condition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by WNV infection or an overt symptom ofpathological processes mediated by WNV infection. The specific amountthat is therapeutically effective can be readily determined by anordinary medical practitioner, and may vary depending on factors knownin the art, such as, for example, the type of pathological processesmediated by WNV infection, the patient's history and age, the stage ofpathological processes mediated by WNV infection, and the administrationof other anti-pathological processes mediated by WNV infection.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

As described in more detail herein, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an RNA from WNV in a cell or mammal, where the dsRNAincludes an antisense strand having a region of complementarity that iscomplementary to at least a part of an mRNA formed in the expression ofan RNA from WNV, and where the region of complementarity is less than 30nucleotides in length, generally 19-24 nucleotides in length, and wherethe dsRNA, upon contact with a cell expressing the gene from WNV,inhibits expression of WNV RNA by at least 40%. The dsRNA of theinvention can further include one or more single-stranded nucleotideoverhangs.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure. Onestrand of the dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence derived from the sequence of an mRNAformed during the expression of an RNA from WNV. The other strand of thedsRNA (the sense strand) includes a region that is complementary to theantisense strand, such that the two strands hybridize and form a duplexstructure when combined under suitable conditions. Generally, the duplexstructure is between 15 and 30, or between 25 and 30, or between 18 and25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 basepairs in length. In one embodiment the duplex is 19 base pairs inlength. In another embodiment the duplex is 21 base pairs in length.When two different siRNAs are used in combination, the duplex lengthscan be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

In further embodiments, the dsRNA includes at least one nucleotidesequence selected from the sequences provided in Table 2. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesof Table 2 and the antisense strand is one of the antisense sequences ofTable 2. Alternative antisense agents that target elsewhere in thetarget sequences provided in Table 2 can readily be determined using thetarget sequence and the flanking WNV sequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Table 2, the dsRNAs featured in the invention can include at leastone strand of a length described herein. It can be reasonably expectedthat shorter dsRNAs having one of the sequences of Table 2 minus only afew nucleotides on one or both ends may be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a partialsequence of at least 15, 16, 17, 18, 19, 20, 21, or 22 or morecontiguous nucleotides from one of the sequences of Table 2, anddiffering in their ability to inhibit the expression of an RNA from WNVin an assay as described herein by not more than 5, 10, 15, 20, 25, or30% inhibition from a dsRNA having the full sequence, are contemplatedby the invention. Further, dsRNAs that cleave within the target sequenceprovided in Table 2 can readily be made using WNV sequence and thetarget sequence provided.

In addition, the dsRNAs provided in Table 2 identify a site in WNV mRNAthat is susceptible to dsRNA based cleavage. As such the presentinvention further includes dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein asecond dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a secondagent will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 2 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in an RNA from WNV. For example, the last 15 nucleotides of SEQID NO:1 combined with the next 6 nucleotides from the target WNV mRNAproduces a single strand agent of 21 nucleotides that is based on one ofthe sequences provided in Table 2.

A dsRNA targeting an RNA of WNV can contain one or more mismatches tothe target sequence. In one embodiment, the dsRNA contains no more than3 mismatches. If the antisense strand of the dsRNA contains mismatchesto a target sequence, it is preferable that the area of mismatch not belocated in the center of the region of complementarity. If the antisensestrand of the dsRNA contains mismatches to the target sequence, it ispreferable that the mismatch be restricted to 5 nucleotides from eitherend, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′end of the region of complementarity. For example, for a 23 nucleotidedsRNA strand which is complementary to a region of an RNA from WNV, thedsRNA generally does not contain any mismatch within the central 13nucleotides. The methods described herein can be used to determinewhether a dsRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an RNA from WNV. Consideration of theefficacy of dsRNAs with mismatches in inhibiting expression of an RNAfrom WNV is important, especially if the particular region ofcomplementarity in an RNA from WNV is known to have polymorphic sequencevariation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Typically, modified dsRNA backbones that do not include a phosphorusatom therein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other dsRNA mimetics, both the sugar and the internucleoside linkage,i.e., the backbone, of the nucleotide units are replaced with novelgroups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-β-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs featured in the invention involveschemically linking to the dsRNA one or more moieties or conjugates whichenhance the activity, cellular distribution or cellular uptake of thedsRNA. Such moieties include but are not limited to lipid moieties suchas a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA,199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem.Let., 1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded dsRNAs

In another aspects featured in the invention, WNV specific dsRNAmolecules that modulate WNV activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. For example, anAAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Exemplary viral vectors are those derived from AV and AAV. In oneembodiment, the dsRNA featured in the invention is expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector comprising, for example, either the U6 or H1 RNA promoters,or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector featured in the invention may be a eukaryotic RNA polymerase I(e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV earlypromoter or actin promoter or U1 snRNA promoter) or generally RNApolymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g. the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below and persist in target cells. Alternatively,viral vectors can be used that provide for transient expression of dsRNAmolecules. Such vectors can be repeatedly administered as necessary.Once expressed, the dsRNAs bind to target RNA and modulate its functionor expression. Delivery of dsRNA expressing vectors can be systemic,such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a WNV RNA over a period of a week or more are alsocontemplated by the invention. Successful introduction of the vectorsfeatured in the invention into host cells can be monitored using variousknown methods. For example, transient transfection can be monitoredusing a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

WNV specific dsRNA molecules can also be inserted into vectors and usedas gene therapy vectors for human patients. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING DSRNA

In one embodiment, the invention provides pharmaceutical compositionshaving a dsRNA as described herein and a pharmaceutically acceptablecarrier. The pharmaceutical composition having the dsRNA is useful fortreating a disease or disorder associated with the expression oractivity of an RNA from WNV such as a viral infection, e.g.,encephalitis. Such pharmaceutical compositions are formulated based onthe mode of delivery. One example is compositions that are formulatedfor direct delivery into the brain parenchyma, e.g., by infusion intothe brain, such as by continuous pump infusion. In one embodiment, thecomposition is formulated for systemic administration via parenteraldelivery, e.g., by intravenous (IV) delivery.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 1 to 50 mg per kilogram body weight per day.For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg,0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, thedsRNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of thedsRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose. In some embodiments, the dsRNA isadministered daily, weekly, biweekly, or monthly.

The effect of a single dose on WNV levels is long lasting, such thatsubsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals.

The present invention includes pharmaceutical compositions that can bedelivered by injection directly into the brain. The injection can be bystereotactic injection into a particular region of the brain (e.g., thesubstantia nigra, cortex, hippocampus, striatum, or globus pallidus), orthe dsRNA can be delivered into multiple regions of the central nervoussystem (e.g., into multiple regions of the brain, and/or into the spinalcord). The dsRNA can also be delivered into diffuse regions of the brain(e.g., diffuse delivery to the cortex of the brain).

In one embodiment, a dsRNA targeting WNV can be delivered by way of acannula or other delivery device having one end implanted in a tissue,such as the brain, e.g., the substantia nigra, cortex, hippocampus,striatum, corpus callosum or globus pallidus of the brain. The cannulacan be connected to a reservoir of the dsRNA composition. The flow ordelivery can be mediated by a pump, such as an osmotic pump or minipump,such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, apump and reservoir are implanted in an area distant from the tissue,such as into the abdomen, and delivery is effected by a conduit leadingfrom the pump or reservoir to the site of release. Infusion of the dsRNAcomposition into the brain can be over several hours or for severaldays, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery tothe brain are described, for example, in U.S. Pat. Nos. 6,093,180, and5,814,014.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or series of treatments. Estimates of effective dosages and invivo half-lives for individual dsRNAs can be made using conventionalmethodologies or determined on the basis of in vivo testing using anappropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by WNV expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The dsRNAs featured in the invention can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by target gene expression. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

Administration

The present invention also includes methods of administeringpharmaceutical compositions and formulations which include dsRNAcompounds, such as those that target an RNA expressed by a pathogen,such as WNV.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media. Thickeners, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions for use with methods featured in the presentinvention include, but are not limited to, solutions, emulsions, andliposome-containing formulations. These compositions may be generatedfrom a variety of components that include, but are not limited to,preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids.

The pharmaceutical formulations for use with the methods of the presentinvention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general, the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun, 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

SNALPs

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, orother nucleic acid-lipid particle. As used herein, the term “SNALP”refers to a stable nucleic acid-lipid particle, including SPLP.

As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 to about90 nm, and are substantially nontoxic. In addition, the nucleic acidswhen present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof, or a mixture thereof. The cationic lipid may comprisefrom about 20 mol % to about 50 mol % or about 40 mol % of the totallipid present in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described inUnited States provisional patent application No. 61/107,998 filed onOct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula I),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-siRNA formulations are as follows:

cationic lipid/non-cationic lipid//choleseterol/PEG-lipid conjugateCationic Lipid Lipid:iRNA ratio Process SNALP 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG- dimethylaminopropane (DLinDMA) cDMA(57.1/7.1/34.4/1.4) lipid:siRNA~7:1 SNALP-2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG- XTC[1,3]-dioxolane (XTC) cDMA 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGExtrusion [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGExtrusion [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 60/7.5/31/1.5, mixing lipid:siRNA~6:1LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 60/7.5/31/1.5, mixing lipid:siRNA~11:1LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 50/10/38.5/1.5 mixing Lipid:siRNA 10:1LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-In-line di((9Z,12Z)-octadeca-9,12- DMG mixing dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]-dioxol-5-amine Lipid:siRNA 10:1(ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-MC-3/DSPC/Cholesterol/PEG- In-line 6,9,28,31-tetraen-19-yl 4- DMG mixing(dimethylamino)butanoate (MC3) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP121,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG- In-linehydroxydodecyl)amino)ethyl)(2- DMG mixinghydroxydodecyl)amino)ethyl)piperazin- 50/10/38.5/1.51-yl)ethylazanediyl)didodecan-2-ol Lipid:siRNA 10:1 (Tech G1)

Carriers

Certain compositions suitable for intracranial administration alsoincorporate carrier compounds in the formulation. As used herein,“carrier compound” or “carrier” can refer to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate dsRNA in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al.,DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions suitable for intracranial administration mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments featured in the invention provide the use ofpharmaceutical compositions containing (a) one or more antisensecompounds and (b) one or more other antiviral agents that function by anon-antisense mechanism. Examples of such antiviral agents include butare not limited to members of classes of agents including reversetranscriptase inhibitors; protease inhibitors; thymidine kinaseinhibitors; sugar or glycoprotein synthesis inhibitors; structuralprotein synthesis inhibitors; nucleoside analogues; and viral maturationinhibitors. Specific non-limiting examples of anti-virals includenevirapine, delavirdine, efavirenz, saquinavir, ritonavir, ribivirin,vidarabine, indinavir, nelfinavir, amprenavir, zidovudine (AZT),stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC),abacavir, acyclovir, penciclovir, valacyclovir, ganciclovir,1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon and adenine arabinoside. When used withthe compounds featured in the invention, such antiviral agents may beused individually, sequentially, or in combination with one or moreother such antiviral agents. Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids mayalso be combined in compositions. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisenseantiviral agents are also within the scope of this invention. Two ormore combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration individually or as a plurality, asdiscussed above, dsRNAs targeting WNV can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by WNV expression. In any event, the administering physiciancan adjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Methods for Treating Diseases Caused by Expression of an RNA from WNV

The invention relates in particular to the administration of a dsRNA ora pharmaceutical composition prepared therefrom for the treatment orprevention of pathological conditions associated with WNV infection,such as encephalitis. Owing to the inhibitory effect on WNV expression,a dsRNA according to the invention or a pharmaceutical compositionprepared therefrom can enhance the quality of life, particularly for apatient infected with WNV.

The invention also relates to the use of a dsRNA or a pharmaceuticalcomposition thereof for treating WNV infection in combination with otherpharmaceuticals and/or other therapeutic methods, such as with knownpharmaceuticals or known therapeutic methods, such as, for example,those which are currently employed for treating or preventing viralinfection. Exemplary antiviral agents are described elsewhere herein.

The invention can also be practiced by including with a specific dsRNA,in combination with another anti-viral agent, such as any conventionalanti-viral agent. The combination of a specific binding agent with suchother agents can potentiate the anti-viral protocol. Thus, the methodfeatured in the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

Treatment of WNV-induced encephalitis can include, in addition toadministration of a dsRNA featured herein, support for the patient whilethey are not able to perform usual bodily functions. Such treatment caninclude administration of anticonvulsants to control seizures, sedation,administration of fluids, and ventilation.

Methods for Inhibiting Expression of an RNA from WNV

In yet another aspect, the invention provides a method for inhibitingthe expression of an RNA from WNV in a mammal, such as a human. Themethod includes administering a dsRNA to the mammal such that expressionof the target WNV RNA is silenced. Because of their high specificity,the dsRNAs featured in the invention specifically target mRNAs (primaryor processed) of the target WNV RNA. Compositions and methods forinhibiting the expression of the WNV RNA using dsRNAs can be performedas described elsewhere herein.

In one embodiment, the method includes administering a compositioncontaining a dsRNA, where the dsRNA includes a nucleotide sequence thatis complementary to at least a part of the mRNA encoded by WNV, to themammal to be treated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 mmole using a MerMade 192 (BioAutomation, Plano, Tex., US) and 500Å controlled pore glass, prepacked columns (BioAutomation) as solidsupport. RNA and RNA containing 2′-O-methyl nucleotides were generatedby solid phase synthesis employing the corresponding phosphoramiditesand 2′-O-methyl phosphoramidites, respectively (ChemGenes, Wilmington,Mass., USA). These building blocks were incorporated at selected siteswithin the sequence of the oligoribonucleotide chain using standardnucleoside phosphoramidite chemistry such as described in Currentprotocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.),John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkageswere introduced by replacement of the iodine oxidizer solution with asolution of 0.1M DDTT in acetonitrile. Further ancillary reagents wereobtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using SpectraMAX 190spectral photometer (Molecular Devices, Sunnywale, Calif., USA). Doublestranded RNA was generated by mixing an equimolar solution ofcomplementary strands in 1×PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2mM KH₂PO₄, pH 7.4) annealing buffer (Ambion, Austin, Tex., USA), heatedin a heating block at 95° C. for 2 minutes and cooled to roomtemperature over a period of 3-4 hours. The annealed RNA solution wasstored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support was used forRNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water. Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) Aadenosine-5′-phosphate C cytidine-5′-phosphate G guanosine-5′ -phosphateT, dT 2′-deoxy-thymidine-5′-phosphate U uridine-5′-phosphate N anynucleotide (G, A, C, or T) a 2′-O-methyladenosine-5′-phosphate c2′-O-methylcytidine-5′-phosphate g 2′-O-methylguanosine-5′-phosphate u2′-O-methyluridine-5′-phosphate S, sdT phosphorothioate backbone linkage

Example 2 In Vitro Efficacy of siRNAs

In vitro activity of siRNA can be determined using methods known in theart, such as those described in Kumar et al. (2006) PLoS Med. 3:504-515. The Neura2a cells (mouse neuronal cell line, obtained fromATCC, Manassas, Va., US) can be seeded in six-well plates at 1×10⁵ cellsper well for 12-16 hours before transfection. SiRNA transfections can beperformed using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., US),iFect (Neuromics, Bloomington, Minn., US), TranslT-TKO (Minis, Madison,Wis.) or JetSI/DOPE (Avanti Polar Lipids, Alabaster, Ala., US) accordingto manufacturer protocols. After incubation for 6 h, cells can be washedand reincubated in the appropriate buffers, and infected with West NileVirus (WNV strain B956 obtained from ATCC (Manassas, Va., UnitedStates)) 24 h post-transfection. Infection levels can be monitored after72 h by flow cytometry using WNV-envelope-specific monoclonal antibody(Chemicon International, Temecula, Calif., US). Comparison of infectionlevels and/or expression of WNV genes of interest in cells transfactedwith a given siRNA(s) demonstrates the efficicacy of said siRNA(s).

In Vivo Efficacy of siRNAs

Female BALB/c mice (6 weeks old, Harlan Sprague Dawley, Indianapolis,Ind.) can be housed under conditions meeting National Institue of Healthregulations. siRNAs, including unconjugated and scrambled controls andvehicle containing no siRNA, can be administered intracranially (IC)through the bregma (4 mm deep vertically into the brain using a Hamiltonsyringe fitted with 30-gauge needle) at different times before the WNVchallenge. The mice can be subsequently challenged with different dosesof WNV by IC inoculation through the bregma at the same spot asdescribed above.

Total mRNA is extracted from mouse brain by rapid homogenization of thetissue in 4 M guanidinuim isothiocyanate followed by centrifugation overa cesium chloride gradient. RNAs (20-40 μg) are resolved in 1.2% agarosegels containing 1.1% formaldehyde and transferred to nylon membranes.The blots are hybridized with a radiolabelled WNV cDNA probe. Probeshybridized to mRNA transcripts are visualized and quantified using aPhosPhorlmager (Molecular Dynamics). After stripping the blots ofradiolabelled probe, they are reprobed with G3PDH cDNA to confirm equalloading. In addition, freshly isolated brain specimens are used to makesingle-cell suspension and neuronal cell infection detected by flowcytometry. Furthermore, brain tissues are homogenized in 10% HBBS-BSAfollowed by repeated passage through the syringe fitted with a 29-gaugeneedle at least 20 times at 4° C. to release intracellular virus. Thustreated brain tissue are used to determine the LD₅₀ of the virus byinoculating serial dilutions of the brain lysate into groups of mice asdescribed in Mahy, B. W. J. (1985) Virology: A Practical Approach, IRLPress, Oxford (United Kingdom).

Example 3

The following sequences targeting West Nile virus mRNA were synthesized:

TABLE 2 Total 25 duplexes Modifications: lower case = 2′-O-methyl s =phosphothiorate SEQ  Exo+ endo light SEQ Alter- ID PAGE or HPLC purification ID  nate AL# NO: sense (5′-3′) AL# NO:antisense (5′-3′) name A-31525  1 AAAAcAcAAcGGAAuGcGAdTsdT A-31526  2UCGcAUUCCGUUGUGUUUUdTsdT A-31527  3 cAAuAuGcuAAAAcGcGGudTsdT A-31528  4ACCGCGUUUuAGcAuAUUGdTsdT si2 A-31529  5 ucAAuAuGcuAAAAcGcGGdTsdT A-31530 6 CCGCGUUUuAGcAuAUUGAdTsdT A-31531  7 cGAuuGGAuGGAuGcuAGGdTsdT A-31532 8 CCuAGcAUCcAUCcAAUCGdTsdT A-31533  9 uGucAAuAuGcuAAAAcGcdTsdT A-3153410 GCGUUUuAGcAuAUUGAcAdTsdT A-31535 11 cuGuGAcAuuGGAGAGucAdTsdT A-3153612 UGACUCUCcAAUGUcAcAGdTsdT si6 A-31537 13 cGGcAuAcAGcuucAAcuGdTsdTA-31538 14 cAGUUGAAGCUGuAUGCCGdTsdT Si7 A-31539 15GuuuGGAAAGGGGAGcAuudTsdT A-31540 16 AAUGCUCCCCUUUCcAAACdTsdT A-31541 17AAuGuccuGGAucAcAcAGdTsdT A-31542 18 CUGUGUGAUCcAGGAcAUUdTsdT A-31543 19uGuuuGGAAAGGGGAGcAudTsdT A-31544 20 AUGCUCCCCUUUCcAAAcAdTsdT A-31545 21GucAAuAuGcuAAAAcGcGdTsdT A-31546 22 CGCGUUUuAGcAuAUUGACdTsdT si11A-31547 23 uuGucAAuAuGcuAAAAcGdTsdT A-31548 24 CGUUUuAGcAuAUUGAcAAdTsdTsi12 A-31549 25 AAGGGGAGcAuuGAcAcAudTsdT A-31550 26AUGUGUcAAUGCUCCCCUUdTsdT A-31551 27 AuGcccuGAAcAccuucAcdTsdT A-31552 28GUGAAGGUGUUcAGGGcAUdTsdT A-31553 29 uuGGAAAGGGGAGcAuuGAdTsdT A-31554 30UcAAUGCUCCCCUUUCcAAdTsdT A-31555 31 uuuGGAAAGGGGAGcAuuGdTsdT A-31556 32cAAUGCUCCCCUUUCcAAAdTsdT A-31557 33 GAAAGGGGAGcAuuGAcAcdTsdT A-31558 34GUGUcAAUGCUCCCCUUUCdTsdT A-31559 35 GuccuGGAucAcAcAGGGAdTsdT A-31560 36UCCCUGUGUGAUCcAGGACdTsdT A-31561 37 AAAGGGGAGcAuuGAcAcAdTsdT A-31562 38UGUGUcAAUGCUCCCCUUUdTsdT A-31563 39 GAAuGuccuGGAucAcAcAdTsdT A-31564 40UGUGUGAUCcAGGAcAUUCdTsdT A-31565 41 ucuGuGAcAuuGGAGAGucdTsdT A-31566 42GACUCUCcAAUGUcAcAGAdTsdT A-31567 43 GuuGucAAuAuGcuAAAAcdTsdT A-31568 44GUUUuAGcAuAUUGAcAACdTsdT A-31569 45 GcucuGuGAcAuuGGAGAGdTsdT A-31570 46CUCUCcAAUGUcAcAGAGCdTsdT A-31571 47 uGcucuGuGAcAuuGGAGAdTsdT A-31572 48UCUCcAAUGUcAcAGAGcAdTsdT si24 A-31573 49 cAcAuGAGAuGuAcuGGGudTsdTA-31574 50 ACCcAGuAcAUCUcAUGUGdTsdT

Example 4 Inhibition of WNV in Humans

A human subject is treated with a dsRNA targeted to a WNV gene toinhibit expression of the WNV gene to treat a condition.

A subject in need of treatment is selected or identified. The subjectcan have a WNV infection or WNV associated condition.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an anti-WNV siRNA is administeredto the subject. The dsRNA is formulated as described herein. After aperiod of time following the first dose, e.g., 7 days, 14 days, and 21days, the subject's condition is evaluated, e.g., by measuring viraltiter, patient temperature, etc. This measurement can be accompanied bya measurement of WNV expression in said subject, and/or the products ofthe successful siRNA-targeting of WNV mRNA. Other relevant criteria canalso be measured. The number and strength of doses are adjustedaccording to the subject's needs.

After treatment, the subject's tumor growth rate is lowered relative tothe rate existing prior to the treatment, or relative to the ratemeasured in a similarly afflicted but untreated subject.

Other embodiments are in the claims.

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) for inhibitingthe expression of a West Nile virus (WNV) RNA in a cell, wherein saiddsRNA comprises at least two sequences that are complementary to eachother and wherein a sense strand comprises a first sequence and anantisense strand comprises a second sequence comprising a region ofcomplementarity which is complementary to an mRNA encoding WNV, andwherein said region of complementarity is less than 30 nucleotides inlength and wherein said dsRNA, upon contact with a cell expressing saidWNV, inhibits expression of said WNV RNA.
 2. The dsRNA of claim 1,wherein said first sequence is selected from the group consisting ofthose in Table 2 and said second sequence is selected from the groupconsisting of those in Table
 2. 3. The dsRNA of claim 1, wherein saiddsRNA comprises at least one modified nucleotide.
 4. The dsRNA of claim2, wherein said dsRNA comprises at least one modified nucleotide.
 5. ThedsRNA of claim 3 or 4, wherein said modified nucleotide is chosen fromthe group of: a 2′-O-methyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 6. ThedsRNA of claim 3 or 4, wherein said modified nucleotide is chosen fromthe group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an a basicnucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide,morpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.
 7. The dsRNA of claim 3 or 4, wherein said firstsequence is selected from the group consisting of those in Table 2 andsaid second sequence is selected from the group consisting of those inTable
 2. 8. The dsRNA of claim 6 or 7, wherein said first sequence isselected from the group consisting of those in Table 2 and said secondsequence is selected from the group consisting of those in Table
 2. 9. Acell comprising the dsRNA of claim
 1. 10. A pharmaceutical compositionfor inhibiting the expression of a West Nile virus (WNV) genome in anorganism, comprising a dsRNA and a pharmaceutically acceptable carrier,wherein the dsRNA comprises at least two sequences that arecomplementary to each other and wherein a sense strand comprises a firstsequence and an antisense strand comprises a second sequence comprisinga region of complementarity which is complementary to an mRNA encodingWNV, and wherein said region of complementarity is less than 30nucleotides in length and wherein said dsRNA, upon contact with a cellexpressing said WNV, inhibits expression of said WNV genome.
 11. Thepharmaceutical composition of claim 10, wherein said first sequence ofsaid dsRNA is selected from the group consisting of those in Table 2 andsaid second sequence of said dsRNA is selected from the group consistingof those in Table
 2. 12. The pharmaceutical composition of claim 10,further comprising a second antiviral agent.
 13. A method for inhibitingthe expression of an RNA from WNV in a cell, the method comprising: (a)introducing into the cell a double-stranded ribonucleic acid (dsRNA),wherein the dsRNA comprises at least two sequences that arecomplementary to each other and wherein a sense strand comprises a firstsequence and an antisense strand comprises a second sequence comprisinga region of complementarity which is complementary to an mRNA encodingWNV, and wherein said region of complementarity is less than 30nucleotides in length and wherein said dsRNA, upon contact with a cellexpressing said WNV, inhibits expression of said WNV RNA; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of an RNA from WNV, therebyinhibiting expression of an RNA from WNV in the cell.
 14. A method oftreating, preventing or managing pathological processes mediated by WNVexpression comprising administering to a patient in need of suchtreatment, prevention or management a therapeutically orprophylactically effective amount of a dsRNA, wherein the dsRNAcomprises at least two sequences that are complementary to each otherand wherein a sense strand comprises a first sequence and an antisensestrand comprises a second sequence comprising a region ofcomplementarity which is complementary to an mRNA encoding WNV, andwherein said region of complementarity is less than 30 nucleotides inlength and wherein said dsRNA, upon contact with a cell expressing saidWNV, inhibits expression of said WNV mRNA.
 15. A vector for inhibitingthe expression of an RNA encoding a WNV genome in a cell, said vectorcomprising a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of a dsRNA, wherein one of thestrands of said dsRNA is complementary to an mRNA encoding WNV andwherein said dsRNA is less than 30 base pairs in length and wherein saiddsRNA, upon contact with a cell expressing said WNV, inhibits theexpression of said WNV genome.
 16. A cell comprising the vector of claim15.